Many forms of DNA damage originate from intrinsic and extrinsic sources. If unrepaired or repaired in an error- prone manner, these lesions induce the mutations and genome rearrangements characteristic of cancer, aging and degenerative diseases. Two particularly pathological lesions are DNA double stranded DNA breaks (DSBs) and collapsed replication forks. The latter originate from stalled replication associated with dissociation of the replisome from the template, and are particularly problematic on the leading strand. To promote the activation of cell cycle checkpoints, the pausing mechanisms to allow time for repair of these lesions and/or replication restart, ssDNA is generated at these sites in a 5'!3' direction. The resulting ssDNA that remains has an exposed 3'-OH group, and acts as a landing pad for assembly of checkpoint signaling complexes as well as recombination enzymes that promote invasion into the sister chromatid. Using the fission yeast Schizosaccharomyces pombe as a gene and pathway discovery tool, we identified a family of XPG-related nucleases (XRNs) as the long sought after enzymes that are necessary and sufficient for end resection at DSBs. This consists of the long known Rad2/Fen1 and Exo1 enzymes that also function in Okazaki Fragment maturation and various Excision Repair pathways. The newly identified third member of this family is known as the Asteroid nucleases (the name Asteroid derives from the proximity of the gene to the Star locus in Drosophila). These include Ast1 in S. pombe and ASTE1 in humans (there is no Asteroid homolog in Saccharomyces cerevisiae), and ASTE1 is a gene frequently mutated in colon and liver cancers characterized by immune infiltrates, though whether it is a driver or a passenger is not known. We have shown that these nucleases are differentially recruited to DSBs depending on their genomic position, and also depending on the complement of nucleases. Additional studies indicate that the XRNs also function at collapsed replication forks, cooperating with several other enzymes that modulate fork stability and processing. Experiments in this proposal further investigate these phenomena utilizing an armory of new tools to study the processing of these lesions across the genome. We further carry out a thorough analysis of Ast1 to bring the understanding of this conserved enzyme to level commensurate with its long-studied cousins. As these initiating events in DNA damage responses feed into many downstream response pathways, this work has significant impact on the study of the many mechanisms that ensure the integrity of the genome.